Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA325780

Local Sample ID:	K562_100nM_Timecourse_Day4_Porphyrin_2
Subject ID:	SU003103
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Cell Strain Details:	K-562

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Sample Preparation:

Sampleprep ID:	SP003109
Sampleprep Summary:	One million cells from culture were collected via centrifugation, washed with 0.9% NaCl, and resuspended in 150 µl of porphyrin extraction buffer (1:4 ratio of 1.7 M HCl:ACN, 1µM deuteroporphyrin IX (Frontier Scientific, D510-9)) and 0.5 µM isotopically labeled amino acids (Cambridge Isotopes, MSK-A2-1.2)). Samples were vigorously shaken for 20 min at 16ºC in a thermomixer (Eppendorf), sonicated for 10 cycles at 4ºC with 30 sec on and 30 sec off, then incubated at 4ºC for 10 min. Following incubation on ice, samples were centrifuged for 10 minutes at 18,000g to pellet cell debris. The supernatant was collected and 40.5 µl super-saturated MgSO4 and 12 µl 5 M NaCl were added. Samples were vortexed for 30 sec and further shaken for 10 min at 16 ºC in a thermomixer. Finally, a 10 min 10,000 rpm centrifugation was used to separate the organic layer (upper) from the aqueous layer (lower). The upper organic layer was collected.

Sampleprep ID:

SP003109

Sampleprep Summary:

One million cells from culture were collected via centrifugation, washed with 0.9% NaCl, and resuspended in 150 µl of porphyrin extraction buffer (1:4 ratio of 1.7 M HCl:ACN, 1µM deuteroporphyrin IX (Frontier Scientific, D510-9)) and 0.5 µM isotopically labeled amino acids (Cambridge Isotopes, MSK-A2-1.2)). Samples were vigorously shaken for 20 min at 16ºC in a thermomixer (Eppendorf), sonicated for 10 cycles at 4ºC with 30 sec on and 30 sec off, then incubated at 4ºC for 10 min. Following incubation on ice, samples were centrifuged for 10 minutes at 18,000g to pellet cell debris. The supernatant was collected and 40.5 µl super-saturated MgSO4 and 12 µl 5 M NaCl were added. Samples were vortexed for 30 sec and further shaken for 10 min at 16 ºC in a thermomixer. Finally, a 10 min 10,000 rpm centrifugation was used to separate the organic layer (upper) from the aqueous layer (lower). The upper organic layer was collected.